We have previously described the isolation of EMS-induced CEM mutants which express intermediate levels of surface CD4 (50-75% of parental line), yet demonstrate markedly reduced susceptibility to HIV-1 infection (multiple strains), as determined by syncytia formation and reverse transcriptase (RT) assays. Quantitative PCR analyses indicated that both viral entry and viral expression are significantly reduced. In order to understand the underlying cellular "defects" which protect them from HIV- 1, we conducted multiple biochem-ical and molecular biology assays.It was found that these mutants respond abnormally to activators of Protein Kinase C (PKC) such as TPA and TNF-alpha. Unlike the parental line, they cannot be induced by TPA to express IL2R and CD3 receptors. TPA induced phosphorylation ofmembrane receptors was seen with some (CD4, CD5), but not other (CD7) receptors. Total PKC enzymatic activity, and PKC translocation occurred normally. In contrast, many assays designed to measure the ability of these mutants to translocate the DNA binding protein NFkB to the nucleous following PKC activation were negative. No activation of HIV-1 LTR-CAT was seen after activation with TPA or TNF- alpha.Furthermore, TAT transactivation of the HIV-1 LTR is also abnormal in these mutants. Recent experiments indicate that the total cytoplasmic NFkB protein is reduced in these mutants, and that following cell activation, there is no induction of NFkB mRNA. Further analyses of these mutants will identify cellular genes which are important for optimal HIV-1 infection and are dependent on the PKC phosphorylation pathway.